Heterotrimeric G proteins transduce signals that mediate the physiological and behavioral effects of many neurotransmitters and Pharmaceuticals. Regulators of G protein signaling (RGS) proteins shorten the duration of G protein signaling by stimulating G protein GTPase activity, but it is still unclear whether the many RGS proteins stimulate specific G proteins in vivo. In addition, the proteins that may modify RGS protein activity or sub-cellular localization are largely unknown. The goal of this proposal is to use biochemistry to understand more about the molecular mechanisms of RGS function in vivo. The first aim is to determine whether RGS proteins target specific G proteins. Western blot analysis of affinity purified RGS protein complexes in C. elegans extracts, paired with GTPase assays, will determine whether two C. elegans RGS proteins, EAT-16 and EGL-10, act exclusively on particular target G proteins, as predicted by genetic studies. The second aim will test whether RGS proteins function as a member of a G protein heterotrimer. The presence of unactivated Ga-beta-RGS heterotrimers, determined by Western blot analysis of affinity purified complexes, would elegantly explain many genetic observations, and more importantly would identify a new role for RGS proteins in G protein signaling. The final goal is to identify other proteins in RGS complexes by mass spectrometry of affinity purified RGS complexes, and to study their role in signaling by phenotypic analysis of mutants.